Date Published: June 30, 2017
Publisher: Public Library of Science
Author(s): Dandan Yang, Zhifeng Zhang, Shaoshuai Liang, Qiankun Yang, Yingrui Wang, Zhenkui Qin, Bin He.
Krüppel-like factor 4 (KLF4) is a kind of zinc finger transcription factor, which is involved in terminal differentiation of epithelial cells and reprogramming of somatic cells to induced pluripotent stem (iPS) cells in mammals. In the present study, we identified a full-length cDNA of Klf4 in Zhikong scallop Chamys farreri (Cf-Klf4) and found that Cf-Klf4 presented a sexual dimorphic expression characteristic in C. farreri gonads. Cf-Klf4 expression was significantly higher in testes than in ovaries from growing stage to mature stage detected by quantitative real-time PCR, and was located in male gametes, except for spermatozoa during spermatogenesis through in situ hybridization and immunohistochemistry, while no positive signal was visible in female gametes during oogenesis. Furthermore, the knockdown of Cf-Klf4 in testes by means of in vivo RNA interference led to an obviously developmental retardance, lower gonadosomatic index, less male gametes and more apoptotic spermatocytes. Interestingly, we found that two out of eight scallops showed a hermaphroditic phenotype characteristic of male-to-female sex reversal when the Klf4 mRNA and protein levels were knocked down in males. These results verified that Klf4 plays an important role in testis functional maintenance and is necessary in spermatogenesis of C. farreri.
Krüppel-like factor 4 (KLF4), a gut-enriched Krüppel-like factor (GKLF) or epithelial zinc finger (EZF), was firstly identified from the mouse serum-deprivation fibroblast cell cDNA library . It has the highly conversed domains of KLF family, the DNA-binding domains with three classical Cys2/His2 zinc fingers at the C-terminus binding GC-box or CACCC-box of the target DNA [2, 3], and two Krüppel-links, TGEKP(Y/F) X which connect zinc fingers . The distinctions between KLF4 and other KLF members are some functional domains, including transcriptional activation domain, transcriptional repression domain, nuclear localization signal (NLS), and a potential PEST domain in mammals [5, 6]. These specific functional domains enable KLF4 to work as transcription regulators. Segre, et al.  and Katz, et al.  reported that losing of KLF4 leads to the deficiency of skin barrier and the reduction of goblet cells, indicating its indispensability in epidermal differentiation. During tumorigenesis, KLF4 works as a crucial transcription factor playing distinctive roles, such as in gastrointestinal and lung cancers, as a tumor suppressor, it regulates the expression of cell cycle-related genes , induces cell apoptosis  or inhibits the activity of telomerase ; or as an oncogene, it suppresses the cdh1/skp2/p27 pathway to promote cell proliferation in breast cancer [11, 12]. Moreover, KLF4 is also an important reprogramme factor which can induce some somatic cells to generate the induced pluripotent stem (iPS) cells [13, 14].
In this study, we firstly reported the characteristics of Cf-Klf4 sexual dimorphic expression in C. farreri testes and ovaries, which is different from mammals. Furthermore, the Klf4 knock-down in C. farreri testes led to the retardance of spermatogenesis, even male-to-female sex reversal, indicating its essential roles in spermatogenesis.
In the present work, we identified a Klf4 full-length cDNA of 2,610 bp in C. farreri. The Cf-Klf4 expression presents a sexually dimorphic characteristic during gametogenesis, and is specifically visible in spermatogonia, spermatocytes and spermatids of C. farreri testes, which is different from that in human beings and mice. Furthermore, the Cf-Klf4 should participate in the regulation of early spermatogenesis and play an important role in maintaining the testis function based on the Cf-Klf4 RNAi analysis. In the future, it remains to study molecular mechanism of Klf4 regulating spermatogenesis in shellfish.